Pharmacopuncture is a new form of acupuncture treatment combining acupuncture and herbal medicine. While the existing acupuncture treatment incorporates physical stimulation of associated meridians and acupoints, pharmacopuncture adds chemical stimulation to the existing acupuncture treatment .
The animals used in this study were 6 week old Sprague-Dawley (SD) rats. The reason SD rats were chosen is that they have generally been used in stability tests of medicine, so the data obtained in this study should be easily compared with many other databases. The mean weights of the rats were 185.1 - 205.9 g and 149.7 - 167.2 g for the male and the female rats, respectively. For all animals, a visual inspection was done, and all animals were weighed using a CP3202S system scale (Sartorius, Germany). After 7 days of acclimatization, the rats general symptoms and changes in weight were observed. The weights were recorded on the last day of acclimatization. No abnormalities were noted. The temperature of the lab was 21.1 - 24.1°C, and the humidity was 40.7% - 64.5%. Sufficient food (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C) and ultra violet (UV)-filtered water were provided. Group separations were done after 7 days of acclimatization. The animals were randomly distributed into 4 groups of 5 male and 5 female rats per group (Table 1): the control, low dose, mid dose and high dose groups.
The expected dose of
The general symptoms (types of toxic symptoms, revealing times, recovery times, etc.) and the mortality were examined 30 minutes, and 1, 2, 4 and 6 hours after the injection on the day of dosing (day 0). From the 1st day to the 14th day of treatment, the general symptoms were examined once a day. The body weights were measured immediately before treatment and at 3, 7 and 14 days after injections.
All animals were fasted for more than 18 hours before the necropsy. The rats were anesthetized by using isoflurane, and blood samples were taken from the abdominal aorta (15 days after injection). An automatic hematology analyzer (ADVIA 2120i, SIEMENS, Germany) was used to analyze blood for the hematological examinations (Table 2). Two mL blood samples were centrifuged for the blood coagulation test (3,000 rpm, 10 minutes). Coagulation test results were measured by using an automated coagulation analyzer (Coapresta 2000, SEKISUI, Japan) (Table 3). For the biochemical tests, the blood remaining after carrying out the hematological tests was centrifuged at 3,000 rpm for 10 minutes, and the serum was collected. Biochemical test results were measured by using an automatic analyzer (7180, HITACHI, Japan) and an electrolyte analyzer (AVL9181, Roche, Germany) (Table 4).
After the observations, organs and tissues from the entire bodies of all surviving animals were visually inspected. Tissue samples of all the animals were fixed in 10% neutral buffered formalin. Routine histological methods, like trimming, dehydration, and paraffin embedding, were conducted on the fixed organs and tissues. Fixed samples were sliced and stained with hematoxylin & eosin (H&E).
All the results from the experiments were analyzed by using statistical analysis system (SAS) software (version 9.3, SAS Institute Inc., U.S.A.). A Bartlett test was conducted to evaluate the homogeneity of the variance and the significance . The one-way analysis of variation (ANOVA) test was conducted when homogeneity of the variance was recognized, and the Dunnett's
During the observation, no deaths or abnormal symptoms occurred in any of the rats in the experimental (0.125, 0.25, 0.5 mL/animal) and the control groups.
No significant changes in body weight were observed in any of the groups (Table 5, Figs. 1,2). No significant changes in the hematological test results were observed in any of the groups, except that the blood urea nitrogen (BUN) of G2 males and the total cholesterol (T-Chol) of G3 males showed statistically significant changes; however, those results had no toxicological meaning because they were not dose dependent (Table 6). No significant changes in the biochemical test results were observed in any of the groups (Table 7), and no abnormalities were noted when visual inspections of all the animals were conducted. No significant changes were noted in the local tolerance test on the injection sites, except for G3 males; however, that result was due to spontaneous generation and had no toxicological meaning because it was not dose dependent.
Pharmacopuncture is the combination of pharmacology and acupuncture. It is useful for the treatment or prevention of several diseases . Pharmacopuncture and herbal acupuncture were introduced to optimize the benefits of acupuncture and herbal medicine by injecting minute quantities of herbs, medicines, blood, oxygen, and allergens into acupoints .
SD rats have been widely used in safety tests of drugs because they have consistent reactions to drugs . Therefore, they are considered to be appropriate for use as test animals.
This study was performed to provide objective safety data for
Further studies should be conducted to evaluate the toxicity more precisely for clinical applications. We need to study the acute and the chronic side effects of
The objective of this study was to analyze the single-dose toxicity of
Groups of animals
[Table. 2] Hematologic examination
[Table. 4] Blood chemical test
Blood chemical test
Body weights (g)
[Table. 6] Mean hematology parameters
Mean hematology parameters
[Table. 7] Mean clinical chemistry
Mean clinical chemistry